5-azacytidine, a nucleoside analogue, is used in the treatment of acute myelogenous leukemia and has been found to cause differentiation of cells in tissue culture and in animals. The latter effect is believed to be due to the inhibition of DNA methylation. We are studying the mechanism of this drug effect on DNA methylases. We have demonstrated that 5-azacytidine inhibits E. coli K12 DNA(cytosine-5)methylase when added to growing cells. It does not affect the DNA(adenine-N6)methylase present in these same cells. The drug inhibits cytosine methylation irreversibly in vivo. DNA extracted from treated cells, which contains 5-azacytosine, inhibits several DNA(cytosine-5)methylases when tested in vitro. The inhibition has the characteristics of a site-specific irreversible inhibitor. This proposal is aimed at identifying the methylase-inhibitor complex and characterizing its structure. To study the specificity of the effects of azacytidine on DNA methylases we will prepare DNA containing azacytosine in unique positions. These substrates will be tested as inhibitors of bacterial and eukaryotic cytosine-dependent DNA methylases. Our objective is to study the DNA-methylase interaction as a means of understanding protein DNA interactions and the mechanism of the methylase reaction. We will use the azacytosine-containing substrates that we will prepare to determine the number of different methylases present in eukaryotic cells and characterize their mechanistic properties. The drug is bactericidal to E. coli B containing EcoRII restriction-modification enzymes dependent on cytosine methylation of DNA, but not to cells not containing such plasmids. The bactericidal effect is due to the presence of the methylase since 1. restrictionless mutants are killed by drug treatment and 2. a methylase mutant survives drug treatment. The basis for this bactericidal effect will be studied by 1. testing other cytosine methylases cloned in E. coli to determine if 5-azacytidine will kill cells containing these methylases, 2. determining if the effect is dependent on methylase levels present in the cell, 3. determining if the drug causes enzyme, in vivo, to complex irreversibly with DNA and 4. determining which repair processes correct these lesions in DNA. We will determine if the basis for the lethal effect of the drug is the same in eukaryotes as it is in procaryotes.